Recent studies suggest that glial cells in the central nervous system (CNS) actively participate in the neural network. Most of the previous studies on the functions of glia have focused on astrocytes identified by the presence of glial filaments. However, in the mature CNS, there exists another major macroglial cells type, the NG2-expressing glia (NG2 cells) which comprise at least 10% of the cell population. Morphological, immunological, and electrophysiological studies indicate that NG2 cells are distinct from astrocytes, mature oligodendrocytes, and microglia. NG2 cells have the potential to differentiate into mature oligodendrocytes in vitro and in vivo and are hence referred to as oligodendrocyte progenitor cells. However, the persistence of a large number of NG2 cells throughout the gray and white matter of adult CNS raises the possibility that they carry out functions besides generating myelinating oligodendrocytes. Based on the ability of purified NG2 proteoglycan to inhibit axonal growth and cause growth cones to collapse, it has been speculated that NG2 cells also play a negative role in axonal growth. On the contrary, preliminary results generated in the Pi's laboratory indicate that growing axons extensively contact NG2 cells and do not immediately collapse upon encountering NG2 cells. The goal of this proposal is to determine whether NG2 cells (and not the molecule itself) promote, inhibit, or cause branching of developing, regenerating, and sprouting axons, and whether these effects are altered by the level of NG2 expression. The hypothesis is that NG2 cells promote or inhibit axonal growth depending on the level of NG2 expressed at the surface, which might be regulated by metalloproteinases. The ability of NG2 cells to promote or inhibit axonal growth as a function of NG2 level will be investigated in dissociated culture (aim 1), in developing corpus callosum in vivo and in living slices (aim 2), and in white matter and gray matter lesion models (aim 3). These studies should elucidate the role of NG2 glia in axonal development and regeneration and may lead to novel concepts and design in therapeutic strategies to promote axonal regeneration following injury.